Described herein are high affinity nucleic acid ligands to tenascin-C. Also described herein are methods for identifying and preparing high affinity nucleic acid ligands to tenascin-C. The method used herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Further disclosed are high affinity nucleic acid ligands to tenascin-C. Further disclosed are RNA ligands to tenascin-C. Also included are oligonucleotides containing nucleotide derivatives chemically modified at the 2xe2x80x2-positions of the purines and pyrimidines. Additionally disclosed are RNA ligands to tenascin-C containing 2xe2x80x2-F and 2xe2x80x2OMe modifications. The oligonucleotides of the present invention are useful as diagnostic and/or therapeutic agents.
Tenascin-C is a 1.1-1.5 million Da, hexameric glycoprotein that is located primarily in the extracellular matrix. Tenascin-C is expressed during embryogenesis, wound healing, and neoplasia, suggesting a role for this protein in tissue remodeling (Erickson and Bourdon, (1989) Ann Rev Cell Biol 5:71-92). Neoplastic processes also involve tissue remodeling, and tenascin-C is over-expressed in many tumor types including carcinomas of the lung, breast. prostate, and colon, astrocytomas, glioblastomas, melanomas, and sarcomas (Soini et at., (1993) Am J Clin Pathol 100(2):145-50; Koukoulis et al., (1991) Hum Pathol 22(7):636-43; Borsi et al., (1992) Int J Cancer 52(5):688-92; Koukoulis et al., (1993) J Submicrosc Cytol Pathol 25(2):285-95; Ibrahim et al., (1993) Hum Pathol 24(9):982-9; Riedi et al., (1998) Dis Colon Rectum 41(1):86-92: Tuominen and Kallioinen (1994) J Cutan Pathol 21(5):424-9; Natal; et al., (1990) Int J Cancer 46(4):586-90; Zagzag et al., (1995) Cancer Res 55(4):907-14; Hasegawa et al., (1997) Acta Neuropathol (Berl) 93(5):431-7; Saxon et al., (1997) Pediatr Pathol Lab Med 17(2):259-66; Hasegawa et al., (1995) Hum Pathol 26(8):838-45). In addition, tenascin-C is overexpressed in hyperproliferative skin diseases, e.g. psoriasis (Schalkwijk et al., (1991) Br J Dermatol 124(1):13-20), and in atherosclerotic lesions (Fukumoto et al., (1998) J Atheroscler Thromb 5(1):29-35; Wallner et al., (1999) Circulation 99(10):1284-9). Radiolabeled antibodies that bind tenascin-C are used for imaging and therapy of tumors in clinical settings (Paganelli et al., (1999) Eur Nucl Med 26(4):348-57; Paganelli et al., (1994) Eur J Nucl Med 21(4):314-21; Bigner et al., (1998) J Clin Oncol 16(6):2202-12; Merlo et al., (1997) Int J Cancer 71(5):810-6).
Aptamers against tenascin-C have potential utility for cancer diagnosis and therapy, as well as for diagnosis and therapy of atheroslerosis and therapy of psoriasis. Relative to antibodies, aptamers are small (7-20 kDa), clear very rapidly from blood, and are chemically synthesized. Rapid blood clearance is important for in vivo diagnostic imaging, where blood levels are a primary determinant of background that obscures an image. Rapid blood clearance may also be important in therapy, where blood levels may contribute to toxicity. SELEX technology allows rapid aptamer isolation, and chemical synthesis enables facile and site-specific conjugation of aptamers to a variety of inert and bioactive molecules. An aptamer to tenascin-C would therefore be useful for tumor therapy or in vivo or ex vivo diagnostic imaging and/or for delivering a variety of therapeutic agents complexed with the tenascin-C nucleic acid ligand for treatment of disease conditions in which tenascin-C is expressed.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. Pat. No. 5,475,096 entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d each of which is specifically incorporated by reference herein in its entirety. Each of these applications, collectively referred to herein as the SELEX patent applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which have the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets in the SELEX method. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d now abandoned, U.S. Pat. No. 5,763,177 entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEXxe2x80x9d and U.S. patent application Ser. No. 09/093,293, filed Jun. 8 1998, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEXxe2x80x9d describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737 entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985 entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d now abandoned, describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d and U.S. Pat. No. 5,683,867 entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow lo the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled xe2x80x9cNucleic Acid Ligand Complexesxe2x80x9d. Each of the above described patents and applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
The present invention describes a method for isolating nucleic acid ligands that bind to tenascin-C with high specificity. Further described herein are nucleic acid ligands to tenascin-C. Also described herein are high affinity RNA ligands to tenascin-C. Further described are 2xe2x80x2fluoro-modified pyrimidine and 2xe2x80x2OMe-modified purine RNA ligands to tenascin-C. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Included herein are the ligands that are shown in Tables 3 and 4 and FIG. 2.
Further included in this invention is a method for detecting the presence of a disease that is expressing tenascin-C in a biological tissue that may contain the disease. Still further included in this invention is a method for detecting the presence of a tumor that is expressing tenascin-C in a biological tissue that may contain the tumor. Further included in this invention is a complex for use in in vivo or ex vivo diagnostics. Still further included in this invention is a method for delivering therapeutic agents for the treatment or prophylaxis of diseased tissues that express tenascin-C. Still further included in this invention is a complex for use in delivering therapeutic agents for treatment or prophylaxis of diseased tissues that express tenascin-C.